PROJECT SUMMARY. Immunological surveillance at barrier surfaces is essential to provide defense against enteric pathogens and maintain mucosal homeostasis. Disruption of the balance between pro-inflammatory and regulatory immune response can lead to a loss of mucosal tolerance and development of chronic inflammatory disease, such as Crohn's disease (CD). The majority of inflammatory responses in CD have been attributed to both innate and adaptive immune cells located in the lamina propria; however, relatively little is known regarding the contribution of the intraepithelial lymphocyte (IEL) compartment to CD pathogenesis. IELs, which include intraepithelial T cells (IET) and intraepithelial ILCs (IE-ILC), contribute to the maintenance of epithelial barrier integrity and function as first line of defense against luminal microorganisms. Due to the difficulty in obtaining a sufficient number of IELs from patient biopsies for in-depth functional analyses, we propose to leverage advances in single-cell RNA sequencing technology and RNA imaging to profile the phenotypic and functional characteristics of ileal IEL subpopulations in health and acute CD. Using transcriptomics as a guide to identify molecular signatures for individual IEL subsets, we will develop single-molecule fluorescence in situ hybridization (sm-FISH) probe multiplexes to visualize these cell populations in patient biopsies. In collaboration with the IBD Genetics Consortium, we will obtain tissue sections from longitudinal endoscopic biopsies of a CD patient cohort that has undergone ileal resection and determine the extent to which IEL phenotype, localization and effector function correlate to disease pathogenesis. These experiments will not only provide the first cell atlas of the human ileal IEL compartment, but may also lead to the development of novel prognostic biomarkers or therapeutic interventions for the treatment of CD.